Compositions and methods for treating acne vulgaris

ABSTRACT

Disclosed are compositions, methods of treatment using the compositions and methods of preparing the compositions for the treatment of acne vulgaris. The compositions include succinic acid and an API selected from the group consisting of salicylic acid, azelaic acid, picolinic acid, benzoyl peroxide, antibiotic, retinoid and combinations thereof in a pharmaceutically acceptable preparation. The compositions that include the combination of succinic acid and another API produce improved efficacy in treating acne vulgaris.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 62/338,334 filed on May 18, 2016, and U.S. Provisional ApplicationSer. No. 62/470,130 filed on Mar. 10, 2017, each of which isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

FIELD

The present invention is related to compositions and methods for thetreatment of acne vulgaris and, in particular, to topical compositionsand methods based upon compositions that are combinations of succinicacid and one or more of topical API including retinoid, salicylic acid,azelaic acid, picolinic acid, benzoyl peroxide, or antibiotic, in apharmaceutically acceptable preparation.

INTRODUCTION

Acne is a significant inflammatory skin disorder and it is consideredthe most common skin disorder. There are four disease factors associatedwith acne vulgaris. They include sebum overproduction, follicularhyperkeratinization, Propionibacterium acnes (P. acnes) proliferation inhair follicles, and inflammation. Acne affects about 50 million peoplein the US. It is highly common among adolescents, affecting up to 95% ofmen and 83% of women in that age group. In approximately 10% of cases,acne has been reported to become severe nodulocystic acne with thepotential of producing lifelong disfiguring scars. Acne lesionstypically recur for years. Therefore, acne is considered a chronicdisease.

The psychosocial morbidity associated with acne is important andincludes depression and suicidal thoughts. In addition to impact onpatient's quality of life, the economic burden of acne treatment issignificant.

Currently available treatments for acne are insufficient. Treatmentoptions against acne vulgaris typically only address one or two diseasefactors—P. acnes proliferation and follicular hyperkeratinization.Treatment options for acne vulgaris suffer from significant side effectsand none provides complete protection. Potential side effects includeskin irritation including burning, erythema, peeling, dryness, andcontact allergy. Antibiotics pose various side effects that range fromcolitis, vaginal candidiasis, and photosensitivity to the development ofbacterial resistance and cross-resistance.

Thus, there is a need to develop new therapies with reduced side effectsand therapies that address multiple acne vulgaris disease factors P.acnes proliferation, follicular hyperkeratinization, and inflammation.

SUMMARY

Accordingly, the applicants herein have succeeded in devising newformulations and methods for treating acne vulgaris.

Thus, in various embodiments, the present invention includes a topicalcomposition for treating acne vulgaris in which the composition includesa combination of succinic acid and salicylic acid in a pharmaceuticallyacceptable preparation. The combination of succinic acid and salicylicacid acts in a synergistic manner in killing Propionibacterium acnes (P.acnes) and treating acne vulgaris, requires ¼ of the dose of succinicacid and salicylic acid to be effective in killing P. acnes and treatacne vulgaris and it is faster acting compared to succinic acid orsalicylic acid alone. In various other embodiments, the presentinvention includes a topical composition for treating acne vulgaris inwhich the composition includes a combination of succinic acid and an APIthat may be topical retinoid, salicylic acid, azelaic acid, picolinicacid, benzoyl peroxide, antibiotics or combinations thereof, in apharmaceutically acceptable preparation. The combination of succinicacid and BPO, succinic acid and azelaic acid, and succinic acid andpicolinic acid acts in additive manner against P. acnes and requires ½of the dose of individual components to kill P. acnes and treat acnevulgaris. The combination of succinic acid and antibiotic preventsantibiotic resistance development and is effective in treating acnevulgaris associated with antibiotic resistant P. acnes. Any combinationscontaining succinic acid prevent P. acnes biofilm development infollicles.

In various other embodiments, the present invention includes a method oftreating acne vulgaris. The method includes administering to a subjectin need thereof, a topical composition comprising a combination ofsuccinic acid and another API, such as retinoid, salicylic acid, azelaicacid, picolinic acid, benzoyl peroxide, or antibiotic, in apharmaceutically acceptable preparation.

In various additional embodiments, the present invention includes amethod of preparing a topical formulation for treatment of acnevulgaris. The method includes combining succinic acid with another APIin a pharmaceutically acceptable preparation, wherein the combination ofsuccinic acid and another API produces additional effect in treatingacne vulgaris, including synergistic or additive effect against P.acnes, prevention of antibiotic resistance development, prevention of P.acnes biofilm formation, or faster acting pharmaceutical composition.

In various embodiments, the present invention includes a topicalcomposition for treating acne vulgaris. The composition may includesuccinic acid and an API that may be topical retinoid, salicylic acid,azelaic acid, picolinic acid, benzoyl peroxide, antibiotics orcombinations thereof, in a pharmaceutically acceptable preparation.

In various other embodiments, the present invention includes a method oftreating acne vulgaris. The method includes administer to a subject inneed thereof, a topical composition that may include succinic acid andan API selected from the group consisting of topical retinoids,salicylic acid, azelaic acid, picolinic acid, benzoyl peroxide,clindamycin and combinations thereof in a pharmaceutically acceptablepreparation.

In various other embodiments, the present invention includes a method oftreating inflammatory acne vulgaris that contains a topical compositionthat includes succinic acid and another component API selected from thegroup consisting of topical retinoids, salicylic acid, azelaic acid,picolinic acid, benzoyl peroxide, clindamycin and combinations thereofin a pharmaceutically acceptable preparation.

In various additional embodiments, the present invention includes amethod of preparing a topical formulation for treatment of acnevulgaris. The method includes combining succinic acid and an APIselected from the group consisting of retinoids, salicylic acid, azelaicacid, picolinic acid, benzoyl peroxide, antibiotics, and combinationsthereof in a pharmaceutically acceptable preparation.

In various embodiments, the compositions and preparations above can beprovided to a subject in a pad, tube, and any container suitable for thedelivery of the compositions and preparations so a subject.

These and other features, aspects and advantages of the presentteachings will become better understood with reference to the followingdescription, examples and appended claims.

DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1 illustrates checkerboard assay using P. acnes for concentrations(mM) of succinic acid and salicylic acid used in each well in which theboxes highlighted in yellow (in the applications from which thisapplication depends; C8 and H3 here) are the individual MIC values foreach API; the green box (in the applications from which this applicationdepends; C3) indicates where the concentrations of the individual MICvalues intersect; the orange box (in the applications from which thisapplication depends; E5) indicates the new MIC value obtained by thecombination of succinic acid and salicylic acid; the boxes shaded ingrey indicate bacterial growth; and the white boxes indicate no growth.

FIG. 2 illustrates the ratios of salicylic acid to succinic aciddetermined by checkerboard assay using P. acnes in which the boxeshighlighted in yellow (in the applications from which this applicationdepends; C8 and H3 here) are the individual MIC values for each API; thegreen box (in the applications from which this application depends; C3)indicates where the concentrations of the individual MIC values for eachAPI intersect; the orange box (in the applications from which thisapplication depends; E5) indicates the new MIC value obtained by thecombination of succinic acid and salicylic acid; the boxes shaded ingrey indicate bacterial growth; and the white boxes indicate no growth.

FIG. 3 illustrates a time-kill assay comparing succinic acidindividually to succinic acid and salicylic acid combination in whicheach plate shows four dilutions for each time point starting with 1:10(right) and ending with 1:10⁴ (left).

FIG. 4 illustrates checkerboard assay using P. acnes for concentrations(mM) of succinic acid and azelaic acid used in each well in which theboxes highlighted in yellow (in the applications from which thisapplication depends; H3 and C8 here) are the individual MIC values foreach API; the green box (in the applications from which this applicationdepends; C3) indicates where the concentrations of the individual MICvalues intersect; the orange box (in the applications from which thisapplication depends; D4) indicates the new MIC value obtained by thecombination of succinic acid and azelaic acid; the boxes shaded in greyindicate bacterial growth; and the white boxes indicate no growth.

FIG. 5 illustrates the ratios of azelaic acid to succinic aciddetermined by checkerboard assay using P. acnes in which the boxeshighlighted in yellow (in the applications from which this applicationdepends; H3 and C8 here) are the individual MIC values for each API; thegreen box (in the applications from which this application depends; C3)indicates where the concentrations of the individual MIC valuesintersect; the orange box (in the applications from which thisapplication depends; D4) indicates the new MIC value obtained by thecombination of succinic acid and azelaic acid; the boxes shaded in greyindicate bacterial growth; and the white boxes indicate no growth.

FIG. 6 illustrates checkerboard assay using P. acnes for concentrations(mM) of succinic acid and picolinic acid used in each well in which theboxes highlighted in yellow (in the applications from which thisapplication depends; H3 and C8 here) are the individual MIC values foreach API; the green box (in the applications from which this applicationdepends; C3) indicates where the concentrations of the individual MICvalues intersect and the orange box (in the applications from which thisapplication depends; D4) indicates the new MIC value obtained by thecombination of succinic acid and picolinic acid. The boxes shaded ingrey indicate bacterial growth and white boxes indicate no growth.

FIG. 7 illustrates the ratios of picolinic acid to succinic aciddetermined by checkerboard assay in which the boxes highlighted inyellow (in the applications from which this application depends; H3 andC8 here) are the individual MIC values for each drug; the green box (inthe applications from which this application depends; C3) indicateswhere the concentrations of the individual MIC values intersect; theorange box (in the applications from which this application depends; D4)indicates the new MIC value obtained by the combination of succinic acidand picolinic acid; the boxes shaded in grey indicate bacterial growth;and the white boxes indicate no growth.

FIG. 8 illustrates checkerboard assay using P. acnes for concentrations(mM) of succinic acid and benzoyl peroxide used in each well in whichthe boxes highlighted in yellow (in the applications from which thisapplication depends; H2 and C8 here) are the individual MIC values foreach API; the green box (in the applications from which this applicationdepends; C2) indicates where the concentrations of the individual MICvalues intersect; the orange box (in the applications from which thisapplication depends; D3) indicates the new MIC value obtained by thecombination of succinic acid and benzoyl peroxide; the boxes shaded ingrey indicate bacterial growth; and the white boxes indicate no growth.

FIG. 9 illustrates the ratios of benzoyl peroxide to succinic aciddetermined by checkerboard assay in which the boxes highlighted inyellow (in the applications from which this application depends H2 andC8 here) are the individual MIC values for each API; the green box (inthe applications from which this application depends; C2) indicateswhere the concentrations of the individual MIC values intersect; theorange box (in the applications from which this application depends; D3)indicates the new MIC value obtained by the combination of succinic acidand benzoyl peroxide; the boxes shaded in grey indicate bacterialgrowth; and the white boxes indicate no growth.

FIG. 10. illustrates checkerboard assay using P. acnes forconcentrations (mM) of succinic acid and clindamycin used in each wellin which the boxes highlighted in yellow (in the applications from whichthis application depends; H3 and C8 here) are the individual MIC valuesfor each API; the green box (in the applications from which thisapplication depends; C3) indicates where the concentrations of theindividual MIC values intersect and the orange box indicates the new MICvalue obtained by the combination of succinic acid and clindamycin; theboxes shaded in grey indicate bacterial growth; and the white boxesindicate no growth.

FIG. 11 illustrates the ratios of clindamycin to succinic aciddetermined by checkerboard assay in which the boxes highlighted inyellow (in the applications from which this application depends; H3 andC8 here) are the individual MIC values for each API; the green box (inthe applications from which this application depends; C3) indicateswhere the concentrations of the individual MIC values intersect; theboxes shaded in grey indicate bacterial growth; and the white boxesindicate no growth.

FIG. 12 illustrates that succinic acid kills antibiotic resistant P.acnes strains in assay in which succinic acid was tested against P.acnes strains RMA #20660 (black) and RMA #20661 (grey) with resistanceto erythromycin, clindamycin, minocycline and doxycycline: wherein theDRM1 (succinic acid) MIC value (determined at 15 mM for each strain) wasdefined as ≥90% decrease in bacterial growth as monitored at an opticaldensity of 600 nm and the results reported as the mean of threeindividual experiments where ND means not detected and clindamycinshowed no ability to inhibit RMA #20660 or RMA #20661 strains.

FIG. 13 illustrates that P. acnes strains RMA #20660 and RMA #20661 butnot P. acnes strain 6919 were resistant to clindamycin.

FIG. 14 shows no resistance development in the presence of DRM1(succinic acid).

FIG. 15 shows that Formulation 1 and Formulation 2 inhibit P. acnesgrowth.

FIG. 16 shows that Formulation 1 and Formulation 2 are bactericidalagainst P. acnes.

FIG. 17 shows production of SE Microbiome Complex by S. epidermidisfermentation with selected prebiotic compounds.

FIG. 18 shows healing of acne in a human subject after applicationFormulation 1.

DETAILED DESCRIPTION

The present invention includes compositions and methods directed to thetreatment of skin diseases, and in particular, inflammatory acnevulgaris. The compositions as well as treatment and formulation methodsare based upon combinations of agents with antimicrobial,anti-hyperkeratinization, anti-comedogenic, and anti-inflammatoryeffects that are effective in treating acne vulgaris.

Abbreviations and Definitions

To facilitate understanding of the invention, a number of terms andabbreviations as used herein are defined below as follows:

As used herein, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a formulation” includes a plurality of suchformulations and reference to “the method” includes reference to one ormore methods and equivalents thereof known to those skilled in the art,and so forth.

As used herein, the term “about” is intended to refer to a range ofvalues above and below a stated value such as for example, valuesencompassing 10% below up to 10% above a stated value.

The term “and/or” is intended to mean either or both of two recitedelements.

“Active pharmaceutical ingredient” (“API”) refers to a substance, inparticular an antimicrobial agent, in a pharmaceutical composition thatis delivered for a desired effect.

As used herein, the term “combination” with respect to active agentsrefers to a composition of two or more active agents, in particular,agent with antimicrobial and/or anti-inflammatory and/oranti-hyperkeratinization and anti-commedogenic effects. In the presentinvention, a combination of active agents may include succinic acid andan API that may include retinoid, salicylic acid, azelaic acid,picolinic acid, benzoyl peroxide, antibiotic, and combinations of two ormore API's.

Reference herein to an API including, but not limited to succinic acid,retinoids, salicylic acid, azelaic acid, picolinic acid, benzoylperoxide and antibiotics is intended to include pharmaceuticallyacceptable solvates, salts, hydrates or hydrated salts, their opticalisomers, racemates, diastereomers, enantiomers or the polymorphiccrystalline structures of the compounds.

In the Examples below, DRM1 refers to succinic acid.

The term “pharmaceutical composition” or ‘pharmaceutical preparation”refers to a composition that combines one or more API's with apharmaceutically acceptable carrier such that the composition issuitable for therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any suitable pharmaceutical carriers, such as a phosphatebuffered saline solution, water, and emulsions, such as an oil/water orwater/oil emulsion, various types of wetting agents and the like. Thecompositions also can include stabilizers and preservatives. Examples ofcarriers, stabilizers and adjuvants, can be found in Remington: TheScience and Practice of Pharmacy, Lippincott Williams & Wilkins,Twenty-First edition (May 19, 2005).

A number of carrier systems have been developed, in particular fortopical application, including by way of non-limiting examples,vesicular systems such as liposomes, niosomes, ethosomes andtransfersomes. (Arora, S. et al., Dermal delivery of drugs usingdifferent, vesicular carriers: A comparative review, J Pharm. 2012;6:237-44).

One such vesicular carrier system is the niosome. As used herein, theterm noisome refers to unilamellar or multilamellar vesicles in which anaqueous phase is encapsulated in highly ordered bilayer made up ofnonionic surfactant (Vyas, J. et al., “Development of topical niosomalgel of benzoyl peroxide,” International Journal of Nanotechnology, vol.2011, Article ID 503158, 6 pages, 2011.). They are nonionic surfactantvesicles by which skin penetration and accumulation are increased in thesuperficial skin strata (Manconi, M. et al., Niosomes as carriers fortretinoin: III. A study into the in vitro cutaneous delivery ofvesicle-incorporated tretinoin, International Journal of Pharmaceutics,2006 311(1-2):11-19.); (Vyas, A. et al., Carrier-Based Drug DeliverySystem for Treatment of Acne, The Scientific World Journal, 2014 Feb. 9;2014:276260. doi: 10.1155/2014/276260. eCollection 2014.)

The term “synergy” as used herein is intended to refer the interactionof discrete antimicrobial agents in which the total antimicrobial effectis greater than the sum of the effects of the antimicrobial agent alone.Conversely, the term “antagonism” is intended to refer to an interactionof antimicrobial agents in which the total antimicrobial effect is lessthan the sum of the effects of the antimicrobial agents alone.

The term “checkerboard experimental design”, is intended to refer to anin vitro two-dimensional array of serial concentrations of testcompounds evaluated in paired or triple combinations for their affect ininhibiting the growth of a target microorganism, measured as a “minimalinhibitory concentration” (MIC), i.e. the lowest concentration thatprevents of inhibits microbial growth. It is used as the basis forcalculation of a “fractional inhibitory concentration index”.

The term “fractional inhibitory concentration index” (FICI) refers to avalue that compares the ratio of inhibitory concentration of acombination with that of individual agents separately. The formula usedto calculate FICI is as follows:FICI=MICA/B/MICA alone+MICB/A/MICB alone

The FIC Index (FICI) value is used to determine whether pairedcombinations of antimicrobial agents can exert inhibitory effectsagainst tested organism that are more or less than the sum of theireffects alone. In theory, FICI<1.0 indicates synergy and FICI>1.0)indicates antagonism. However, a more conservative calculation has beenrecommended such that FICI data may be interpreted as “synergy” for anFICI≤0.5, “antagonism” for an FICI>4.0 and “no interaction” interactionfor an FICI>0.5-4.0. (Odds, F. C., Synergy, antagonism, and what thechequerboard puts between them, Journal of Antimicrobial Chemotherapy(2003) 52, 1. As used herein, the FICI is interpreted as follows:FICI≤0.5=synergyFICI>0.5−≤1=additive effectFICI>1−≤4=indifferenceFICI>4=antagonism

Unless otherwise indicated, concentrations are given as mass weightpercentages, i.e. w/w %. Mass weight percentages (w/w %) for combinationformulations are calculated as follows:mass % a=mass(a)÷(mass(a)+mass(b)+mass(c)+ . . . )×100 (w/w %).

Compositions

The present invention includes compositions and treatment methods basedupon compositions that are combinations of agents having antimicrobial,anti-inflammatory, anti-hyperkeratinization, and anti-commedogenicactivities. Such compositions include succinic acid and an API selectedfrom the group consisting of topical retinoids, salicylic acid, azelaicacid, picolinic acid, benzoyl peroxide, antibiotics and combinationsthereof.

In various embodiments, the amount of succinic acid in the compositionmay be from about 0.1 to about 10 w/w % and in particular, from about0.1, about 0.2, about 0.5 or about 0.75, about 1 w/w % to about 2, about3, about 4, about 5, about 7.5 or about 10 w/w %. In variousembodiments, the amount of succinic acid in the composition may be about0.1, about 0.2, about 0.5, about 0.75, about 1, about 2, about 3, about4, about 5, about 6, about 7, about 8, about 9 or about 10 w/w %. Thesuccinic acid is combined with one or more API's.

In various embodiments, salicylic acid may be included in thecomposition. Salicylic may be present in the composition in an amount offrom about 0.05 to about 5 w/w % and in particular, from about 0.05,about 0.1, about 0.2, about 0.5 or about 0.75 w/w % to about 1, about 2,about 3, about 4 or about 5 w/w %. In various embodiments, the amount ofsalicylic acid in the composition may be about 0.05, about 0.1, about0.2, about 0.5, about 0.75, about 1, about 2, about 3, about 4 or about5 w/w %.

In various embodiments, azelaic acid may be included in the composition.Azelaic acid may be present in the composition in an amount of fromabout 0.05 to about 10 w/w % and in particular, from about 0.05, about0.1, about 0.2, about 0.5 or about 0.75, about 1 w/w % up to about 2,about 3, about 4, about 5, about 7.5 or about 10 w/w %. In variousembodiments, the amount of azelaic acid in the composition may be about0.05, about 0.1, about 0.2, about 0.5, about 0.75, about 1, about 2,about 3, about 4 about 5, about 7.5 or about 10 w/w %

In various embodiments, picolinic acid may be included in thecomposition. Picolinic acid may be present in the composition in anamount of from about 0.01 to about 10 w/w % and in particular, fromabout 0.01, about 0.02, about 0.05, about 0.1, about 0.2 or about 0.5w/w % to about 0.75, about 1, about 2, about 3, about 4, about 5, about7.5 or about 10 w/w %. In various embodiments, the amount of picolinicacid in the composition may be about 0.01, about 0.02, about, about0.05, about 0.1, about 0.2, about 0.5, about 0.75, about 1, about 2,about 3, about 4, about 5, about 7.5 or about 10 w/w %

In various embodiments, benzoyl peroxide may be included in thecomposition. Benzoyl peroxide may present in the composition in anamount of from about 0.01 to about 5 w/w % and in particular, from about0.01, about 0.02, about 0.05, about 0.1, about 0.2 or about 0.5 w/w % toabout 0.75, about 1, about 2, about 3, about 4 or about 5 w/w %. Invarious embodiments, the amount of benzoyl peroxide in the compositionmay be about 0.01, about 0.02, about, about 0.05, about 0.1, about 0.2,about 0.5, about 0.75, about 1, about 2, about 3, about 4 or about 5 w/w%

In various embodiments, antibiotic may be included in the composition.The antibiotic includes erythromycin, clindamycin, minocycline anddoxycycline, or any other antibiotic with activity against P. acnes.Antibiotic may be present in the composition in an amount of from about0.001 up to about 2 w/w % and in particular, from about 0.001, about0.002, about 0.005 or about 0.0075, about 0.01, about 0.02, about 0.03,about 0.04, about 0.05 w/w % up to about 0.075, about 0.1, about 0.2,about 0.3, about 0.4, about 0.5 about 1 or about 2% w/w %. In variousembodiments, the amount of antibiotic in the composition may be about0.001, about 0.002, about 0.005, about 0.0075, about 0.01, about 0.02,about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08,about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about1% or about 2 w/w %.

The compositions of the present invention may be incorporated into apharmaceutically acceptable carrier system which may include creams,gels, lotions or other types of suspensions that can stabilize thecombination of agents and deliver them to the affected area of skin bytopical applications. Pharmaceutically acceptable carrier systems mayalso contain ingredients that include, but are not limited to, saline,aqueous electrolyte solutions, ethanol, dimethyl sulfoxide, dimethylisosorbide, isopropyl myristate, lauryl lactate, diisopropyl adipate,sodium lauryl sulfoacetate; ionic and nonionic osmotic agents such assodium chloride, potassium chloride, glycerol, propylene glycol anddextrose; pH adjusters and buffers such as salts of hydroxide,phosphate, citrate, acetate, borate; and trolamine; antioxidants such assalts, acids and/or bases of bisulfite, sulfite, metabisulfite,thiosulfite, ascorbic acid, acetyl cysteine, cystein, glutathione,butylated hydroxyanisole, butylated hydroxytoluene, tocopherols, andascorbyl palmitate; compounds such as lecithin, phospholipids; petroleumderivatives such as mineral oil and white petrolatum; fats such aslanolin, peanut oil, palm oil, soybean oil; mono-, di-, andtriglycerides; polymers of acrylic acid such as carboxypolymethylenegel, and hydrophobically modified cross-linked acrylate copolymer;polysaccharides such as dextrans and glycosaminoglycans such as sodiumhyaluronate. Such pharmaceutically acceptable carriers may be preservedagainst bacterial contamination using preservatives, including, but arenot limited to, benzalkonium chloride, ethylene diamine tetra-aceticacid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol,methylparaben, thimerosal, and phenylethyl alcohol, or may be formulatedas a non-preserved formulation for either single or multiple use.

Methods of Treatment

The methods of treatment of the present invention are useful for thetreatment of skin diseases including acne vulgaris and inflammatory acnevulgaris. Treatment of acne vulgaris may be by topically administeringto a subject a composition that includes a combination of active agents.

The compositions of the present invention can be administered at avariety of intervals. In some instances, administration may be once aday. In other instances, administration can be less or more frequently,such as 1, 2, 3, or 4 times a day, 1 time every 2 days, or once a week.

The treatment methods may be monitored by following any of thepathogenic aspects of acne vulgaris including number of acne lesions,sebum excretion, follicular keratinization, comedone formation,bacterial colonization of the follicle, P. acnes proliferation and/orinflammation.

EXAMPLES

Aspects of the present teachings may be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way. In the examples below, DRM1refers to succinic acid.

Example 1—this Example Illustrates the Antimicrobial Effect ofCombinations of DRM1 (Succinic Acid) and Salicylic Acid Using aCheckerboard Assay Design

Bacterial Cultivation

P. acnes (ATCC 6919) was cultured on Brucella plates supplemented with0.1 g/L hemin, 0.01 g/L vitamin K, and 5% (v/v) defibrinated sheep'sblood at 37° C. under anaerobic conditions using a Gas-Pak (BD). Foreach assay, an inoculum of P. acnes was prepared by suspending a colonyof P. acnes in 5 mL Reinforced Clostridium medium (RCM) and growing at37° C. and 250 RPM under anaerobic conditions for 30 hours. Cultureswere harvested, washed with PBS and resuspended in fresh, sterile RCMbroth to a concentration of 2×106 CFU/ml.

Minimum Inhibitory Concentration (MIC) Determination

Individual MIC values were determined for each drug according to themicrobroth dilution method. Briefly, 2-fold serial dilutions of eachdrug (50 μl) were added to wells in a 96-well plate followed by 50 μl ofthe prepared P. acnes inoculum as described in Example 1. Media withoutP. acnes and medium with P. acnes only were used for negative andpositive controls, respectively, and contained the highest concentrationof solvent used in each drug analysis. Plates were incubated underanaerobic conditions using a Gas-Pak (BD) for 48 hours at 37° C.Following incubation, each well was resuspended by pipetting and theoptical density at 600 nm (OD600) was determined on a plate reader. TheMIC value was defined as the first well showing ≥90% reduction in growthcompared to controls.

In Vitro Checkerboard Assay

A checkerboard assay was used to determine if combining DRM1 (succinicacid) with commonly used topical treatments for acne showed synergy,indifference or antagonism between the drugs. Checkerboard assays wereperformed in 96 well plates as an 8×8 grid. Two-fold serial dilutions ofAPI A (25 μl) were added at 4× the desired final concentration across 8rows of the microplate. Two-fold serial dilutions of API B (25 μl) wereadded at 4× the desired final concentration to 8 columns on themicroplate so that the highest concentration of API A and the highestconcentration of API B were in the same well at one corner of the 8×8grid diagonal from the corner containing the lowest concentration ofeach drug. For each drug, a set of concentrations greater than and lessthan the MIC as calculated above was used. An inoculum of P. acnes (50μl) as described in example 1 was added to each well containing API andthe plate was incubated at 37° C. for 48 hours under anaerobicconditions and the individual and combined MIC values were determined asstated in example 2 for the MIC assay. The FICI for each combination wasdetermined using the following equation:FICI=MICA/B/MICAalone+MICB/A/MICBalone

Synergy was defined as an FICI≤0.5, additivity was defined as anFICI>0.5≤1.0, indifference was defined as an FICI>1≤4 and antagonism wasdefined as an FICI>4.0.

Checkerboard Combination of DRM1 (Succinic Acid) and Salicylic Acid

Table 1, below, shows the MIC values for Salicylic Acid.

As shown in FIGS. 1 and 2 and Table 1, the combined MIC of the two drugswas found to be 3.75 mM DRM1 (succinic acid) and 1.9 mM salicylic acidas seen in FIG. 1. This corresponded to an FICI of 0.5 suggestingsynergy was occurring between DRM1 (succinic acid) and salicylic acid.Growth inhibition was seen when salicylic acid and DRM1 (succinic acid)were used at a ratio of 1:32 when the concentration of DRM1 (succinicacid) was ≥15 mM and the concentration of salicylic acid was ≥0.47 mM,when salicylic acid and DRM1 (succinic acid) were used in a ratio of 1:8when the concentration of DRM1 (succinic acid) was ≥7.5 mM and theconcentration of salicylic acid was ≥0.94 mM, when salicylic acid andDRM1 (succinic acid) were used in a ratio of 1:2 when the concentrationof DRM1 (succinic acid) was ≥3.75 mM and the concentration of salicylicacid was ≥1.9 mM, when salicylic acid and DRM1 (succinic acid) were usedin a ratio of 2:1 when the concentration of DRM1 (succinic acid) was≥1.9 and the concentration of salicylic acid was ≥3.75 mM, and whensalicylic acid and DRM1 (succinic acid) were used in a ratio of 4:1 whenthe concentration of DRM1 (succinic acid) was ≥0.94 mM and theconcentration of salicylic acid was ≥3.75 mM as seen in FIG. 2. Usingthe control P. acnes strain, ATCC 6919, DRM1 (succinic acid) incombination with SA resulted in an FICI of 0.5 suggesting the two activepharmaceutical ingredients (APIs) are working in synergy. The mostsynergistic concentration of the two APIs occurred at a ratio of 2:1(DRM1:SA) when DRM1 (succinic acid) (3.75 mM) was combined with SA (1.9mM), at a quarter of the MIC concentration required for each componentalone. (See Table 1 below)

TABLE 1 MIC's for Salicylic Acid in combination with Succinic Acid DRM1Synergistic/ Salicylic (Succinic Additive/ Acid Acid Ratio Indifferent/(mM) mM) (Salicylic/DRM1) FICI Antagonist 0 15 — — — 7.5 0 — — — 1.93.75 1:2 1.9/7.5 + 3.75/15 = 0.5 Synergistic ≥0.47 ≥15  1:32 ≥0.47/7.5 +≥ 15/15 = ≥ 1.06 Additive ≥0.94 ≥7.5 1:8 ≥0.94/7.5 + ≥ 7.5/15 = ≥ 0.625Additive ≥1.9 ≥3.75 1:2 ≥1.9/7.5 + ≥ 3.75/15 = ≥ 0.5 Synergistic ≥3.75≥1.9 2:1 ≥3.75/7.5 + ≥ 1.9/15 = ≥ 0.626 Additive ≥3.75 ≥0.94 4:1≥3.75/7.5 + ≥ 0.94/15 = ≥ 0.563 Additive

Example 2—this Example Illustrates the Time-Kill Assay of DRM1 (SuccinicAcid) and Salicylic Acid in Combination

DRM1 (succinic acid) and salicylic acid were used individually atconcentrations 2× their MIC values (30 mM DRM1 (succinic acid) and 15 mMsalicylic acid) and in combination at 0.5×MIC for DRM1 (succinic acid,7.5 mM) and 1×MIC for salicylic acid (7.5 mM) Assays were performed inRCM broth with 12% propylene glycol in 96-well plates. A finalconcentration of 5×105 CFU/ml P. acnes 6919 was used for each reactionand each assay was performed under anaerobic conditions using a Gas-Pakat 37° C. At various time points (0, 15, 30, 60 and 120 minutes) 10 μlof each sample was removed and diluted 1:10 to 1:104 in sterile PBS and20 μl of each dilution was spread on a Brucella broth agar platesupplemented with 0.1 μg/ml hemin and 0.01 μg/ml vitamin K using thetrack plate method. FIG. 3 shows a consistent growth between all threesamples and the control at 0 minutes. After 15 minutes, growth wasevident on the individual DRM1 (succinic acid) and salicylic acidplates; however, no growth was detected on the DRM1 (succinicacid)-salicylic acid combination plate. After 30 minutes of incubation,no growth was detected on any of the plates. The increased speed atwhich the DRM1 (succinic acid)-salicylic acid combination inhibited P.acnes growth suggests the two APIs performed in a synergistic oradditive way.

Example 3—this Example Illustrates the Antimicrobial Effect ofCombinations of DRM1 (Succinic Acid) and Azelaic Acid Using aCheckerboard Assay Design

Bacterial cultivation, MIC calculation and Checkerboard Assay designwere as described in Example 1. Table 2, below, shows MIC values forAzelaic Acid.

As shown in FIGS. 4 and 5 and Table 2, the combined MIC of the two drugswas at 7.5 mM DRM1 (succinic acid) and 3.75 mM azelaic acid as seen inFIG. 4. This corresponded to an FICI of 1.0 suggesting the interactionbetween DRM1 (succinic acid) and azelaic acid was additive. Growthinhibition was seen when azelaic acid and DRM1 (succinic acid) were usedat a ratio 1:32 when the concentration of DRM1 (succinic acid) was ≥15mM and the concentration of azelaic acid was ≥0.47 mM, when azelaic acidand DRM1 (succinic acid) were used in a ratio of 1:8 when theconcentration of DRM1 (succinic acid) was ≥7.5 mM and the concentrationof azelaic acid was ≥0.94 mM, when azelaic acid and DRM1 (succinic acid)were used in a ratio of 1:4 when the concentration of DRM1 (succinicacid) was ≥7.5 mM and the concentration of azelaic acid was ≥1.9 mM,when azelaic acid and DRM1 (succinic acid) were used in a ratio of 1:2when the concentration of DRM1 (succinic acid) was ≥7.5 mM and theconcentration of azelaic acid was ≥3.75 mM, when azelaic acid and DRM1(succinic acid) were used in a ratio of 1:1 when the concentration ofDRM1 (succinic acid) was ≥3.75 and the concentration of azelaic acid was≥3.75 mM, when azelaic acid and DRM1 (succinic acid) were used in aratio of 2:1 when the concentration of DRM1 (succinic acid) was ≥1.9 mMand the concentration of azelaic acid was ≥3.75 mM, and when azelaicacid and DRM1 (succinic acid) were used in a ratio of 8:1 when theconcentration of DRM1 (succinic acid) was ≥0.94 mM and the concentrationof azelaic acid was ≥7.5 mM as seen in FIG. 5. DRM1 (succinic acid) incombination with AZA had additive effect and killed P. acnes at half theconcentration each when used in combination. The concentration of AZAcan be lowered which results in less side effect while maintainingefficacy of the combination.

TABLE 2 MIC's for Azelaic Acid in combination with Succinic Acid DRM1Synergistic/ Azelaic (Succinic Additive/ acid Acid Ratio Indifferent/(mM) mM) (Azelaic/DRM1) FICI Antagonist 0 15 — — — 7.5 0 — — — 3.75 7.51:2 3.75/7.5 + 7.5/15 = 1.0 Additive ≥0.47 ≥15  1:32 ≥0.47/7.5 + ≥ 15/15= ≥ 1.06 Indifferent ≥0.94 ≥7.5 1:8 ≥0.94/7.5 + ≥ 7.5/15 = ≥ 0.625Additive ≥1.9 ≥7.5 1:4 ≥1.9/7.5 + ≥ 7.5/15 = ≥ 0.75 Additive ≥3.75 ≥7.51:2 ≥3.75/7.5 + ≥ 7.5/15 = ≥ 1.0 Additive ≥3.75 ≥3.75 1:1 ≥3.75/7.5 + ≥3.75/15 = ≥ 0.75 Additive ≥3.75 ≥1.9 2:1 ≥3.75/7.5 + ≥ 1.9/15 = ≥ 0.63Additive ≥7.5 ≥0.94 8:1 ≥7.5/7.5 + ≥ 0.94/15 = ≥ 1.06 Indifferent

Example 4—this Example Illustrates the Antimicrobial Effect ofCombinations of DRM1 (Succinic Acid) and Picolinic Acid Using aCheckerboard Assay Design

Bacterial cultivation, MIC calculation and Checkerboard Assay designwere as described in Example 1. Table 3, below, shows MIC values forpicolinic acid.

As shown in FIGS. 6 and 7 and Table 3, DRM1 (succinic acid, 0-60 mM) andpicolinic acid (0-7.5 mM) were added to a 96-well plate in 2-fold serialdilutions and combined with P. acnes 6919 as described in example 1. Thecombined MIC of the two drugs was at 7.5 mM DRM1 (succinic acid) and0.94 mM picolinic acid as seen in FIG. 6. This corresponded to an FICIof 1.0 suggesting the interaction between DRM1 (succinic acid) andpicolinic acid was additive. Growth inhibition was seen when picolinicacid and DRM1 (succinic acid) were used at a ratio 1:128 when theconcentration of DRM1 (succinic acid) was ≥15 mM and the concentrationof picolinic acid was ≥0.12 mM, when picolinic acid and DRM1 (succinicacid) were used in a ratio of 1:64 when the concentration of DRM1(succinic acid) was ≥15 mM and the concentration of picolinic acid was≥0.23 mM, when picolinic acid and DRM1 (succinic acid) were used in aratio of 1:16 when the concentration of DRM1 (succinic acid) was ≥7.5 mMand the concentration of picolinic acid was ≥0.47 mM, when picolinicacid and DRM1 (succinic acid) were used in a ratio of 1:8 when the ofDRM1 (succinic acid) was ≥7.5 mM and the concentration of picolinic acidwas ≥0.94 mM, when picolinic acid and DRM1 (succinic acid) were used ina ratio of 1:4 when the concentration of DRM1 (succinic acid) was ≥3.75mM and the concentration of picolinic acid was ≥0.94 mM, when picolinicacid and DRM1 (succinic acid) were used in a ratio of 1:2 when theconcentration of DRM1 (succinic acid) was ≥3.75 mM and the concentrationof picolinic acid was ≥1.88 mM, when picolinic acid and DRM1 (succinicacid) were used in a ratio of 1:1 when the concentration of DRM1(succinic acid) was ≥1.9 mM and the concentration of picolinic acid was≥1.9 mM and when picolinic acid and DRM1 (succinic acid) were used in aratio of 2:1 when the concentration of DRM1 (succinic acid) was ≥0.9 mMand the concentration of picolinic acid was ≥1.88 mM as seen in FIG. 7.DRM1 (succinic acid) in combination with picolinic acid had additiveeffect and killed P. acnes at half the concentration each when used incombination. The concentration of picolinic acid can be lowered whichresults in less side effect while maintaining efficacy of thecombination.

TABLE 3 MIC's for Picolinic Acid in combination with Succinic Acid DRM1Synergistic/ Picolinic (Succinic Additive/ acid Acid Ratio Indifferent/(mM) mM) (Picolinic/DRM1) FICI Antagonist 0 15 — — — 1.88 0 — — — 0.947.5 1:8 0.94/1.88 + 7.5/15 = 1.0 Additive ≥0.12 ≥15  1:128 ≥0.12/1.88 +≥ 15/15 = ≥ 1.06 Indifferent ≥0.23 ≥15  1:64 ≥0.23/1.88 + ≥ 15/15 = ≥1.12 Indifferent ≥0.47 ≥7.5  1:16 ≥0.47/1.88 + ≥ 7.5/15 = ≥ 0.75Additive ≥0.94 ≥7.5 1:8 ≥0.94/1.88 + ≥ 7.5/15 = ≥ 1.0 Additive ≥0.94≥3.75 1:4 ≥0.94/1.88 + ≥ 3.75/15 = ≥ 0.75 Additive ≥1.88 ≥3.75 1:2≥1.88/1.88 + ≥ 3.75/15 = 1.25 Indifferent ≥1.88 ≥1.9 1:1 ≥1.88/1.88 + ≥1.9/15 = ≥ 1.13 Indifferent ≥1.88 ≥0.9 2:1 ≥1.88/1.88 + ≥ 0.9/15 = ≥1.06 Indifferent

Example 5—this Example Illustrates the Antimicrobial Effect ofCombinations of DRM1 (Succinic Acid) and Benzoyl Peroxide (BPO) Using aCheckerboard Assay Design

Bacterial cultivation, MIC calculation and Checkerboard Assay designwere as described in Example 1. Table 4, below shows MIC values for BPO.

As shown in FIGS. 8 and 9 and Table 4, DRM1 (succinic acid, 0-7085μg/ml) and benzoyl peroxide (0-250 μg/ml) were added to a 96-well platein 2-fold serial dilutions and combined with P. acnes 6919 as describedin example 3. DRM1 (succinic acid) had an individual MIC value of 1771μg/ml and BPO had an individual MIC (succinic acid) value of 125 μg/ml.The combined MIC of the two drugs was at 885.7 μg/ml DRM1 (succinicacid) and 62.5 μg/ml BPO as seen in FIG. 8. This corresponded to an FICIof 1.0 suggesting the interaction between DRM1 (succinic acid) and BPOwas additive. Growth inhibition was seen when BPO and DRM1 (succinicacid) were used at ratios of 1:113, 1:226, and 1:453 when DRM1 (succinicacid) was used at a concentration ≥1771 μg/ml and BPO was used at aconcentration ≥15.6 μg/ml, ≥7.8 μg/ml, and ≥3.9 μg/ml, respectively, asseen in FIG. 9. Growth inhibition was also seen at a ratio of BPO toDRM1 (succinic acid) of about 1:14 and 1:28 when the concentration ofDRM1 (succinic acid) was ≥885.7 μg/ml and BPO was used at ≥62.5 μg/ml,and ≥31.25 μg/ml, respectively, or when the ratio of BPO to DRM1(succinic acid) was about 1:7, 1:3.5, 1:1.8, or 1:0.88 at concentrationsof BPO≥125 μg/ml and concentrations of DRM1 (succinic acid) at ≥885.7μg/ml, ≥442 μg/ml, ≥221 μg/ml, and ≥110 μg/ml, respectively. DRM1(succinic acid) in combination with BPO had additive effect and killedP. acnes at half the concentration each when used in combination. Theconcentration of BPO can be lowered which results in less side effectwhile maintaining efficacy of the combination.

TABLE 4 MIC's for Benzoyl Peroxide (BPO) in combination with SuccinicAcid DRM1 Synergistic/ (Succinic Additive/ BPO Acid Ratio Indifferent/(μg/ml) μg/ml) (BPO/DRM1) FICI Antagonist 0 1771 — — — 125 0 — — — 62.5885.7 1:14 62.5/125 + 885.7/1771 = 1.0 Additive ≥3.9 ≥1771 1:453≥3.9/125 + ≥ 1771/1771 = ≥ 1.03 Indifferent ≥7.8 ≥1771 1:226 ≥7.8/125 +≥ 1771/1771 = ≥ 1.06 Indifferent ≥15.6 ≥1771 1:113, ≥15.6/125 + ≥1771/1771 = ≥ 1.12 Indifferent ≥31.25 ≥885.7 1:28 ≥31.25/125 + ≥885.7/1771 = ≥ 0.75 Additive ≥62.5 ≥885.7 1:14 ≥62.5/125 + ≥ 885.7/1771= ≥ 1.0 -Additive- ≥125 ≥885.7 1:7 ≥125/125 + ≥ 885.7/1771 = ≥ 1.5Indifferent ≥125 ≥442 1:3.5 ≥125/125 + ≥ 442/1771 = 1.25 Indifferent≥125 ≥221 1:1.8 ≥125/125 + ≥ 221/1771 = ≥ 1.12 Indifferent ≥125 ≥1101:0.88 ≥125/125 + ≥ ≥110/1771 = ≥ 1.06 Indifferent

Discussion of Examples: 1-5

Examples 1-5 Illustrate DRM1 (Succinic Acid) Combinations that MaximizeEfficacy and Minimize Side Effects

DRM1 (succinic acid) increases efficacy of other acne treatmentsincluding salicylic acid, benzoyl peroxide, azelaic acid, picolinicacid, retinoids, etc. while reducing side effects of the second agentson the skin. This means that combinations can be used in which theamount of a side effect causing agent (such as benzoyl peroxide (BPO),salicylic acid, azelaic acid, retinoids, etc.) can be reduced whilemaximizing efficacy. We have identified DRM1 (succinic acid) containingcombinations and the ratios between DRM1 (succinic acid) and the secondagent that are optimal for acne treatment. All combinations improve theperformance of the second agent without increasing side effects—theyreduce side effects.). The most efficacious combination seems to be DRM1(succinic acid)+salicylic acid at ratio 2:1. This combination shows thehighest synergistic effects against P. acnes, reduces inflammation(provided by DRM1), and has anti-hyperkeratinization (provided bysalicylic acid) and anti-commedogenic (provided by salicylic acid)properties. This combination addresses three acne vulgaris diseasesfactors. In addition, the combinations result in faster killing of P.acnes (time kill assays in Example 2) and prevents biofilm formation inDRM1 concentration above 7.5 mM (Example 8). The combinations of DRM1(succinic acid) with a second agent can be optimized using specificmolar ratios to achieve optimal efficacy while minimizing side effects.In addition to treating acne vulgaris in all patients includinginflammatory acne vulgaris, DRM1 (succinic acid) containing combinationscould be particularly useful in patients who are prone to side effectson the skin and would benefit from suing topical treatments that containreduced amount of BPO, salicylic acid, azelaic acid, retinoids, etc.

Example 6—this Example Illustrates the Antimicrobial Effect ofCombinations of DRM1 (Succinic Acid) and Clindamycin Using aCheckerboard Assay Design

Bacterial cultivation, MIC calculation and Checkerboard Assay designwere as described in Example 1. Table 5, below, shows MIC values forBPO.

As shown in FIGS. 10 and 11 and Table 5, DRM1 (succinic acid, 0-7085μg/ml) and clindamycin (0-0.25 μg/ml) were added to a 96-well plate in2-fold serial dilutions and combined with P. acnes 6919 as described inexample 3. The combined MIC of the two drugs was at 1771 μg/ml DRM1(succinic acid) and 0.06 μg/ml clindamycin, which were the same MICvalues for each individual treatment as seen in FIG. 10. Thiscorresponded to an FICI of 2.0 suggesting the interaction between DRM1(succinic acid) and clindamycin was indifferent with no indication of anantagonistic interaction. Growth inhibition was seen when clindamycinand DRM1 (succinic acid) were used at ratios of 1:28341, 1:56683,1:113366, 1:226732, 1:453465 when DRM1 (succinic acid) was used at aconcentration ≥1771 μg/ml and clindamycin was used at a concentration≥0.063 μg/ml, ≥0.031 μg/ml, ≥0.016 μg/ml, ≥0.008 μg/ml, and ≥0.004μg/ml, respectively, as seen in FIG. 11.

TABLE 5 MIC's for Clindamycin in combination with Succinic Acid DRM1Synergistic/ (Succinic Ratio Additive/ Clindamycin Acid (Clindamycin/Indifferent/ (μg/ml) μg/ml) DRM1) FICI Antagonist 0 1771 — — — 0.063 0 —— — 0.063 1771 1:28341 0.063/0.063 + 885.7/1771 = 2.0 Indifferent ≥0.004≥1771 1:453465 ≥0.004/0.063 + ≥ 1771/1771 = ≥ 1.07 Indifferent ≥0.008≥1771 1:226732, ≥0.008/0.063 + ≥ 1771/1771 = ≥ 1.13 Indifferent ≥0.016≥1771 1:113366 ≥0.016/0.063 + ≥ 1771/1771 = ≥ 1.27 Indifferent ≥0.031≥1771 1:56683, ≥0.031/0.063 + ≥ 885.7/1771 = ≥ 1.52 Indifferent ≥0.063≥1771 1:28341 ≥0.063/0.063 + ≥ 885.7/1771 = ≥ 2.05 Indifferent ≥0.063≥221.4 1:3514 ≥0.063/0.063 + ≥ 221.4/1771 = ≥ 1.17 Indifferent ≥0.063≥110.7 1:1757 ≥0.063/0.063 + ≥ 110.7/1771 = ≥ 1.11 Indifferent

Table 6 summarizes the MIC values from Examples 1, 3, 4, 5 and 6.

TABLE 6 Summary of MIC values DRM1 Synergistic/ Conc. Ratio Additive/API (Succinic (API/ Indifferent/ API Conc. Acid) DRM1) FICI AntagonistSalicylic  1.9 mM 3.75 mM 1:2 1.9/7.5 + 3.75/15 = 0.5 Synergistic AcidAzelaic 3.75 mM  7.5 mM 1:2 3.75/7.5 + 7.5/15 = 1.0 Additive acid  Picolinic 0.94 mM  7.5 mM 1:8 0.94/1.88 + 7.5/15 = 1.0 Additive AcidBenzoyl 62.5 885.7 1:14 62.5/125 + 885.7/1771 = 1.0 Additive Peroxideμg/ml μg/ml Clindamy 0.063 1771 1:28341 0.063/0.063 + Indifferent cinμg/ml μg/ml 885.7/1771 = 2.0

Example 7—this Example Shows that DRM1 (Succinic Acid) Killed P. acnesStrains that are Resistant to Antibiotics

The MIC values for DRM1 (succinic acid) against multi-resistant P. acnesstrains were determined as described in example 1. Briefly, P. acnesstrains (RMA #20660 and RMA #20661) with low susceptibility toerythromycin, clindamycin, minocycline, and doxycycline were incubatedwith DRM1 (succinic acid, 0-120 mM) and the minimum inhibitoryconcentration was determined. As illustrated in FIGS. 12 and 13, DRM1(succinic acid) was found to be bactericidal againstantibiotic-resistant P. acnes strains RMA #20660 and RMA #20661. The MICfor DRM1 (succinic acid) against multi-resistant P. acnes strains was 15mM. This was similar to the MIC value for non-resistant strains of P.acnes demonstrating that DRM1 (succinic acid) is able to kill P. acnesstrains resistant to commonly used antibiotics.

Example 8—this Example Shows that DRM1 (Succinic Acid) Prevents BiofilmFormation

DRM1 (succinic acid) prevented biofilm formation in biofilm-formingstrains at a concentration of 7.5 mM and above. Biofilm formation is animportant factor in P. acnes pathogenicity and biofilm is detected inapproximately half of hair follicles of acne patients.

Example 9—this Example Illustrates that DRM1 (Succinic Acid) PreventsResistance Development in P. acnes Strains

Resistance development may be measured by two methods: single step forthe determination of spontaneous frequency of resistance development andserial passage for the determination of progressive resistancedevelopment. Single step resistance frequency was determined byspreading a large inoculum of P. acnes (10⁹ to 10¹⁰ CFU) onto a bloodagar plate containing 4×, 8×, 16×, or 32× the MIC for DRM1 (succinicacid) as described in Example 2. The spontaneous resistance frequencywas calculated from the number of colonies that grow on platescontaining DRM1 vs. the number of colonies that grow on DRM1-free agar.As shown in FIG. 14, no colonies grew and therefore no resistance wasdetected. For progressive resistance selection, P. acnes (106 CFU/ml)will be exposed to a series of tubes containing serial 2-fold increasingconcentrations of DRM1 (succinic acid) for a total of 10 cycles (72hours each). The MIC of DRM1 (succinic acid) will be determined from thetubes containing the highest drug concentration. Tubes with the highestdrug concentration that still shows turbidity will be used to inoculate(100 μl) a new series of tubes containing drug dilutions. To investigatewhether DRM1 (succinic acid) could prevent the emergence of antibioticresistance, experiments will be performed as described in the presenceof 0.25 times the MIC of DRM1 (succinic acid). The resistance stabilityof each clone will be determined by sub-culturing onto drug-free agarfor three cycles and determining the DRM1 (succinic acid) MIC via themicrobroth dilution method as described in Example 1.

P. acnes strains did not develop spontaneous or progressive resistanceto DRM1 (succinic acid) and including DRM1 (succinic acid) incombination with antibiotics for acne treatment prevents antibioticresistance emergence and development.

Discussion of Examples: 6-9

Examples 6-9 illustrate that combinations with antibiotics preventsresistance development while reducing side effects.

DRM1 (succinic acid) combined with topical antibiotics can be used toreplace side effect causing BPO in combinations containing antibioticsand BPO. DRM1 (succinic acid) is bactericidal. Many antibiotics arebacteriostatic which promotes resistance development. DRM1 (succinicacid) is effective against antibiotic resistant P. acnes strains andprevents emergence of antibiotic resistant P. acnes strains when used incombinations with antibiotics. In addition, DRM1 prevents biofilmformation.

Example 10: Synthetic SE Microbiome Complex

A synthetic SE Microbiome Complex (the “SE Microbiome Complex”)containing a defined combination of S. epidermis metabolites andprebiotics was prepared by combining at least the following in aconcentration of 0-10%:

-   -   1. At least one metabolite produced by S. epidermidis (e.g.,        succinic acid, acetic acid, lactic acid, butyric acid, other        short chain fatty acids, other metabolites including small        molecules, peptides or proteins).    -   2. At least one prebiotic compound (e.g., glycerol, sucrose,        maltose, lactose, other carbohydrate or other compounds that        supports fermentation and growth of S. epidermidis)

The components of the synthetic SE Microbiome Complex were produced bychemical synthesis or by microbial fermentation using wild-type orengineered microorganisms. The SE Microbiome Complex was optimized forbactericidal properties against P. acnes, skin tolerance, and cosmeticacceptance. The SE Microbiome Complex was formulated for use for thetreatment of acne vulgaris.

Example 11: Formulation SE Microbiome Complex with Salicylic Acid forthe Treatment of Acne Vulgaris Using Medicated Acne Treatment Pads

Acne Treatment Pads Formulation (Formulation 1):

The formulation was prepared that contained salicylic acid (1%), organicaloe vera (50%), propanediol (15%), organic grain alcohol (10%) and SEMicrobiome Complex. The SE Microbiome complex consisted of 2% succinicacid, 1% Brij L4, 1% dimethyl isosorbide ether, and 1% glycerin(prebiotic compound). Optionally, organic lemon essential oil was addedto a final concentration of 0-1% and grapefruit seed extract to a finalconcentration of 0-1%. Sodium hydroxide was used to adjust the solutionpH to 3.9-4.5. Succinic acid was prepared by microbial fermentation orby chemical synthesis. Alternatively, succinic acid in the formulationwas replaced by conditioned media prepared by fermenting S. epidermidisas follows: S. epidermidis was incubated in phenol red-free rich mediumwith glycerol (20 g/l) for one to six days. After that, microorganismswere discarded by centrifugation at 5,000 g for 30 min. Fermented mediawere then passed through 0.2-μm-pore-size filters. Conditioned mediacontained succinic acid and other S. epidermidis fermentationmetabolites. Alternatively, conditioned media was used in addition tosuccinic acid. Alternatively, glycerin was replaced by other prebioticcompound (such as sucrose, maltose, lactose or other carbohydrates andother prebiotic compounds that support the fermentation of S.epidermidis). Alternatively, organic components in the formulation werereplaced by non-organic equivalent components. The formulated solutionwas poured over pads (material—rayon, Lyocell, Polyester or similar) ina container. The pads were used for applying the topical treatment foracne vulgaris.

Example 12: Formulation of a Salicylic Acid and Succinic AcidCombination for the Acne Spot Treatment of Acne Vulgaris Using aConcentrated Gel

Acne spot treatment formulation (Formulation 2) was prepared containing:Salicylic acid (2%), propanediol (15%), organic grain alcohol (10-20%)and SE Microbiome Complex—super strength (consisting of 4% succinicacid, 3% Brij L4, 5% dimethyl isosorbide). Sodium hydroxide was used toadjust the solution pH to 3.9-4.5. Hydroxyethyl cellulose or eco-gel wasadded to gel the solution at a concentration of 1-3%. Optionally,organic tee tree essential oil (0-10%) and lemon essential oil (0-1%)was added. Optionally, organic aloe vera (0-50%) was added. Optionally,glycerin or other prebiotic compounds (0-5%) were added (such as sucroseor other carbohydrates or prebiotic compounds that support thegrowth/fermentation of S. epidermidis). Alternatively, conditioned mediaof fermenting S. epidermidis (prepared as described in Example 1) wasused instead of or in addition to succinic acid. Alternatively, organiccomponents in the formulation were replaced by non-organic equivalentcomponents. Gel was filled into tubes for application. Gel was used fortopical treatment of acne vulgaris.

Example 13: Bacterial Cultivation

P. acnes (ATCC 6919) was cultured on reinforced Clostridial medium (RCM)agar plates at 37° C. under anaerobic conditions using a Gas-Pak (BD).For each assay, an inoculum of P. acnes was prepared by suspending acolony of P. acnes in 5 mL RCM and growing at 37° C. under anaerobicconditions for 72 hours. Cultures were harvested, washed with PBS andresuspended in fresh, sterile RCM broth to a concentration of 2×10⁶CFU/ml.

Example 14: Formulation 1 and Formulation 2 are Inhibitory Against P.acnes

Formulation 1 (Example 11) and Formulation 2 (Example 12) was incubatedin 96-well plate with P. acnes prepared as described in Example 13.Control sample contained P. acnes in media only. Plates were incubatedunder anaerobic conditions using a Gas-Pak (BD) for 72 hours at 37° C.Following incubation, the plate was examined visually. Control samplesexhibited growth as expected. No growth was observed in wells containingFormulation 1 and Formulation 2 (FIG. 15). The MIC (determined by serialdilutions) was determined to be at 0.015% succinic acid for Formulation1 and Formulation 2.

Example 15: Formulation 1 and Formulation 2 is Bactericidal Against P.acnes

The minimal bactericidal concentration for each formulated drug wasdetermined according to the Clinical Laboratory Standards Institute(CLSI) document M26-A. Sample wells from the MIC assay in Example 14were serially diluted 1:10 to 1:10⁴ in PBS and each dilution (5 μl) wasplated on RCM agar plates.

The plates were incubated at 37° C. in a Gas-Pak. Following incubation,Formulation 1 (from Example 11) and Formulation 2 (from Example 12)exhibited no growth (FIG. 16). Control samples exhibited growth asexpected. MBC concentration was determined as a concentration that showsa ≥3 log₁₀ decrease in bacterial growth. The results showed thatFormulation 1 and Formulation 2 are bactericidal against P. acnes. Usingserial dilutions, the MBC was determined to be 0.015% succinic acid forFormulation 1 and 0.015% succinic acid for Formulation 2.

Example 16: Production of SE Microbiome Complex by S. epidermidisFermentation with Prebiotic Compounds and Testing Against P. acnes

This example illustrates the growth inhibitory effects of S. epidermidisconditioned media prepared with various prebiotics against P. acnes.

Preparation of Prebiotics: Glycerol, sucrose, β-lactose, andD-(+)-maltose were purchased from Sigma-Aldrich. Stock solutions of eachprebiotic (4% w/v) were prepared in water and filtered through a 0.22 μmfilter for sterilization.

Preparation of S. epidermidis conditioned media: S. epidermidis (ATCC12228) was cultured on trypic soy broth (TSB) agar plates at 37° C. Foreach assay, an inoculum of S. epidermidis was prepared by suspending onecolony of S. epidermidis from a TSB agar plate into 7 ml of TSB brothand growing at 37° C., 215 RPM for 16 hours. The overnight culture wassubcultured by adding 100 μl of overnight culture to 10 ml of fresh TSB.Once mid-log phase was reached, the cells were pelleted bycentrifugation, washed with PBS and resuspended in fresh PBS. In 15 mlculture tubes, 4.9 ml of Rich Broth (20 g/L yeast extract, 6 g/L TSB)was combined with 40 μl 0.5% phenol red indicator, 5 ml of 4% prebiotic(glycerol, sucrose, β-lactose, or D-(+)-maltose) or PBS as a control,and S. epidermidis (10⁵ CFU/ml) and placed in an anaerobic Gas-Pak (BD).The cultures were incubated at 37° C., 215 RPM for 6 days. Followingfermentation, the cells were pelleted and the supernatants were filteredthrough a 0.22 μm filter for sterilization.

Minimum Inhibitory Concentration (MIC) Determination: Individual MICvalues were determined for each conditioned medium according to themicrobroth dilution method. P. acnes (ATCC 6919) was cultured in RCMbroth and harvested during mid-log phase of growth. The bacterial pelletwas washed and resuspended in PBS. A stock of P. acnes (10⁷ CFU/ml) wasprepared using the estimation that an OD600 nm of 1.0 corresponds toabout 10⁷ CFU/ml. Two-fold serial dilutions of each conditioned medium(90 μl) in PBS were added to wells in a 96-well plate followed by 10 μlof the prepared P. acnes inoculum. Conditioned media without P. acnesand RCM medium with P. acnes only were used for negative and positivecontrols, respectively. Plates were incubated under anaerobic conditionsusing a Gas-Pak (BD) for 48 hours at 37° C. Following incubation, eachwell was resuspended by pipetting and the optical density at 600 nm(OD600) was determined on a plate reader. The MIC value was defined asthe first well showing ≥90% reduction in growth compared to controls.

Fermentation of prebiotics by S. epidermidis: FIG. 17 shows S.epidermidis cultures grown with (a) PBS, (b) glycerol, (c) β-lactose,(d) maltose, (e) sucrose under anaerobic conditions. After growing forsix days under anaerobic conditions, the S. epidermidis culturescontaining glycerol, sucrose, D-(+)-maltose and β-lactose showed achange of color from red to yellow indicating the acidification of themedia when compared to the PBS control. No change in color occurred inPBS control (a). The acidification of the media was due to the formationof short chain fatty acid (e.g., succinic acid, acetic acid, lacticacid, butyric acid) resulting from the fermentation of prebiotics by S.epidermidis. The glycerol sample was not as bright of a yellow comparedto the other three prebiotics and still had a tint of pink. Thisindicates that sucrose, maltose and galactose prebiotics showed the mostefficient fermentation and highest production of short chain fatty acidsand therefore these are the preferred prebiotics to use in formulationsfor the treatment of acne.

P. acnes growth inhibition by S. epidermidis conditioned media:Conditioned media prepared by fermentation in the presence of prebioticsglycerol, sucrose, D-(+)-maltose and β-lactose under anaerobic conditionwere able to inhibit P. acnes growth (undiluted media was the MIC). RCMcontrol media prepared in the absence of prebiotics were not inhibitory.

Example 17: Skin Tolerability and Cosmetic Acceptance Testing

Formulation 1 and Formulation 2 with and without aloe vera was tested inhuman volunteers for skin tolerability and cosmetic acceptance.Evaluation was performed by using questionnaire and a scoring system.Score of 1-5 was used. Score 1 for skin tolerability corresponded toirritation, skin reddening. Score 5 for skin tolerability correspondedto no adverse reaction and excellent tolerability. Score 1 for cosmeticacceptability corresponded to unacceptable product due to smell andfeel. Score 5 for cosmetic acceptability represented excellent cosmeticacceptability (smell and feel). Commercially available products wereused as controls. Results are recorded in table 1. Results indicate thataddition of aloe vera significantly improves skin tolerability and thatFormulation 1 and Formulation 2 have significantly improved cosmeticacceptance (smell and feel) compared to commercially available products.

TABLE 7 Skin tolerability and cosmetic acceptance scores Skintolerability Cosmetic acceptance Formulation score score Formulation 1with aloe vera 5 5 Formulation 1 without aloe vera 4 4 Commerciallyavailable acne 3 1 treatment pads with 1% salicylic acid Formulation 2 55

Example 18: Testing of Treatments Containing Combinations Products inHuman Subjects

Formulation 1 (Example 11) and Formulation 2 (Example 12) were tested inhuman subjects. The Formulation 1 was applied using Acne Treatment Padsone or two times per day. Improvement in acne symptoms was observed(FIG. 18, panel A vs. panels B and C). Panel A shows acne lesions priorto treatment, panels B and C show disappearance of acne lesions posttreatment with Formulation 1. Formulation 2 (Acne Spot Treatment gel)was used as a spot treatment on individual acne lesions in humansubjects. The subjects reported reduction in pain and inflammation in5-10 minutes post application, significantly faster healing of acnelesions compared to benzoyl peroxide and lack of hyperpigmentation (darkspot side effect observed when benzoyl peroxide was used). When AcneSpot Treatment was used in combination with hydrocolloid Acne PimplePatches, the healing of acne lesions was visible in less than a day.

Example 19: Five Step Acne Treatment System Using Combination of TopicalTreatment Containing SE Microbiome Complex, Oral Probiotics and OralSupplements

The five step treatment system combined the topical treatment containingSE Microbiome Complex with oral probiotics and oral supplements (such aszinc). Oral probiotics contained a diverse combination of probioticstrains of 500 million 30 billion CFUs/capsule. The combination ofprobiotic strains included any combination of Bifidobacterium lactis,Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve,Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillusrhamnosus, Lactobacillus salivarius, Lactobacillus plantarum,Lactobacillus bulgaricus, Lactobacillus casei, Lactococcus lactis, oradditional probiotic strains and optionally contained prebiotics (suchas FOS or Arabinogalactan).

Five step treatment system consisted of:

Step 1: Washing face in the morning and evening.

Step 2: Applying Acne Treatment Pads (Formulation 1 from Example 11) toaffected areas and Acne Spot Treatment (Formulation 2 from Example 12)to individual pimples.

Step 3: Covering whiteheads and inflamed pimples with Acne PimplePatches (containing hydrocolloid) to treat blemishes overnight.

Step 4: Taking Probiotics (1-3 times daily) and 25-50 mg Zinc (1 timedaily with a meal).

Step 5: Repeating for at least 2 months.

Compared to subjects using only topical products, the subjects who usedtopical products in combination with oral probiotics and zinc achievedimprovement in acne symptoms.

Example 20: Combination Products Containing Retinoids

This example will be performed to screen combinations of DRM1 andretinoids (tretinoin, adapalene, tazarotene, isotretinoin, retinylpalmitate, retinol, or retinaldehyde) for antimicrobial andanti-inflammatory properties, and to determine if the compounds candecrease the amount of acne lesions in acne.

The minimal inhibitory concentration (MIC) and minimum bactericidalconcentration of combinations of DRM1 and retinoids will be determinedto monitor the growth inhibitory effects of the combination. Todetermine the MIC values, DRM1 will be analyzed in concentrationsranging from 1-100 mM and the retinoids will vary in concentrations from0.025%-0.1%. Two-fold serial dilutions of the DRM1-retinoid combinationwill be made in PBS (50 μl). An inoculum of 2×10⁶ CFU/ml P. acnes in RCMwill be prepared and 50 μl will be added to the wells containing theDRM1-retinoid combinations.

Bactericidal properties of the DRM1 and retinoid combinations will beanalyzed using the time-kill assay. Briefly, 0.4 ml of 10× the desiredfinal concentration of DRM1 and retinoid will be added to 2.8 ml RCMbroth along with 0.4 ml of 10⁷ CFU/ml P. acnes. Media with P. acnes onlywill be used for a positive control. A separate tube will be preparedfor each time point for all measured concentrations of the DRM1-retinoidcombination. The assay will be performed under anaerobic conditions at37° C. The reaction will be analyzed after 0, 0.5, 1, 2, 4, 8, and 24hours. At each time point the corresponding tube will be removed and thesample will be diluted 1:10 to 1:10⁵ in PBS. Each dilution will bespotted (10 μl) in triplicate on RCM agar plates and incubated underanaerobic conditions using a Gas-Pak (BD) for 4-7 days at 37° C.Following incubation, the plate will be visually examined and thebacterial counts for each time point will be determined.

The best in vitro performing DRM1-retinoid combination, as determined bythe MIC assay and time-kill assay, will be formulated into a topicalcream, gel, or solution applied with pads. The topical formulation willbe applied to acne infected areas twice a day (morning and night)following cleansing of the skin.

It is expected that the application of a DRM1-retinoid combination toacne infected skin will decrease the acne lesion counts when compared tocontrols placebo, DRM1 alone or retinoid alone. It is expected that thecount of both inflammatory and non-inflammatory lesions will decreasecompared to controls.

Other Embodiments

The detailed description set-forth above is provided to aid thoseskilled in the art in practicing the present invention. However, theinvention described and claimed herein is not to be limited in scope bythe specific embodiments herein disclosed because these embodiments areintended as illustration of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description which do not depart from thespirit or scope of the present inventive discovery. Such modificationsare also intended to fall within the scope of the appended claims.

REFERENCES CITED

All publications, patents, patent applications and other referencescited in this application are incorporated herein by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application or other reference wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes. Citation of a reference herein shallnot be construed as an admission that such is prior art to the presentinvention.

OTHER PUBLICATIONS INCORPORATED HEREIN BY REFERENCE IN THEIR ENTIRETYINCLUDE

Wang Y(1), Kuo S, Shu M, Yu J, Huang S, Dai A, Two A, Gallo R L, Huang CM. Staphylococcus epidermidis in the human skin microbiome mediatesfermentation to inhibit the growth of Propionibacterium acnes:implications of probiotics in acne vulgaris. Appl Microbiol Biotechnol.2014 January; 98(1):411-24. doi: 10.1007/s00253-013-5394-8. Epub 2013Nov. 22.

What is claimed is:
 1. A topical composition comprising a combination ofsalicylic acid and succinic acid present in a 1:2 ratio to produce asynergistic effect in treating acne vulgaris.
 2. The composition ofclaim 1, wherein salicylic acid is present at concentration from 0.1 to5%.
 3. The composition of claim 1, wherein succinic acid is present atconcentration from 0.1 to 10%.
 4. A method of treating acne vulgaris,the method comprising administering to a subject in need thereof, thetopical composition of claim
 1. 5. The method of claim 4 furthercomprising: (a) washing patient areas affected by acne in the morningand evening with mild detergent; (b) applying a solution containing SEMicrobiome Complex to the patient areas affected by acne and gelcontaining SE Microbiome Complex to individual acne lesions or pimplesor acne lesions and pimples; (c) optionally covering whiteheads andinflamed acne lesions or pimples on patient with hydrocolloid-containingpatches to treat blemishes overnight; (d) taking probiotics containingLactobacillus and Bifidobacterium (1-3 times daily) and 25-50 mg of Zinc(1 time daily with a meal); and (e) repeating (a)-(d) for at least 2months.
 6. The topical composition of claim 1, further comprising aloevera (0-50 w/w %), propanediol (15 w/w %), grain alcohol (10-20 w/w %),lemon essential oil (0-1 w/w %), grapefruit seed extract (0-1 w/w %),sodium hydroxide to adjust the solution pH to 3.9-4.5, and SE MicrobiomeComplex.
 7. The topical composition of claim 6, wherein the SEMicrobiome complex comprises 0.5-4 w/w % succinic acid, 1-5 w/w %dimethyl isosorbide, and 0-5 w/w % carbohydrate-based prebiotic agent.8. The topical composition of claim 6, further comprising aloe vera. 9.The topical composition of claim 6, the SE Microbiome Complex comprisessterile conditioned media prepared by fermenting S. epidermidis.
 10. Thetopical composition of claim 6, wherein the SE Microbiome Complexcomprises at least one of succinic acid, acetic acid, lactic acid,butyric acid, or a short chain fatty acid, and at least one of glycerol,sucrose, maltose, or lactose.
 11. A pad comprising the topicalcomposition of claim
 1. 12. A container comprising the pads of claim 11.13. The topical composition of claim 6, further comprising: a gellingagent at a concentration of 0.5-3 w/w % to the solution.
 14. The topicalcomposition of claim 13, filled into tubes for application.
 15. A tubecomprising the topical composition of claim 13.